! ABSTRACT Detection of head and neck squamous cell carcinoma (HNSCC) at or close to surgical margins increases the risk of death at 5-years by 90%. However, large surgical margins to ensure clear margins can greatly increase a patient's morbidity with functional and cosmetic damage of oral and facial structures. Although promising, single fluorescence agents targeting epidermal growth factor receptor (EGFR) are qualitative at best, have moderate tumor-to-normal tissue contrast, and have not yet demonstrated long-term improved patient outcomes. Our recently developed in vivo paired-agent imaging method allows quantification of extracellular receptor expression (and identification of cancerous tissue) by utilizing simultaneously delivered targeted and non-targeted (i.e., perfusion) agents to reference the plasma delivery and leakage. The ability to eliminate the non-binding fluorescence contribution in the tumor and surrounding normal tissues increases the observed tumor-to-normal tissue contrast and improves our ability to observe microscopic tissue burden. The central HYPOTHESIS is that EGFR targeted Paired-Agent Imaging for Resection During Surgery (PAIRS) will improve HNSCC patient long-term survival and decrease morbidity. The ultimate RESEARCH GOAL is to demonstrate that PAIRS outperforms both traditional (non-guided) and single, fluorescent-agent guided surgical resection in detecting microscopic tumor in surgical margins of a spontaneous HNSCC patient-surrogate model. Clinical translation of PAIRS will be fully realized by completion of the outlined SPECIFIC AIMS: Aim 1 - Develop clinic-ready integrated FGS system for HNSCC PAIRS under operating room lights; Aim 2 - Optimize the PAIRS model in HNSCC tumors and derive clinical imaging parameters; and Aim 3 - Validate the clinical viability of PAIRS in both murine xenografts and spontaneous, volunteer dog model as compared to traditional and single, fluorescent-agent guided surgical resection. The translational INNOVATION will include production of a fully integrated PAIRS system, and a robust set of clinical imaging parameters. To expedite clinical translation of PAIRS, we will leverage two fluorescent agents currently being used in clinical trials: ABY-029 (a fluorescent anti-EGFR Affibody molecule) and IRDye 700DX. In addition, the clinical SIGNIFICANCE of PAIRS to reduce morbidity and increase survival will have been demonstrated in both the controlled environment of murine xenografts as well as the high variability of spontaneous veterinary cancers in dogs. As a translational biophysical chemist, I have lead the development of the paired-agent model as a tissue identifying technique and have assembled a strong team of notable cancer and clinical imaging researchers. !